α7nAChR激动剂对体外循环致大鼠脑损伤时NLRP3炎症小体的影响

Effect of α7nAChR agonist on NLRP3 inflammasome during brain injury induced by cardiopulmo-nary bypass in rats

目的评价α7烟碱型乙酰胆碱受体(α7nAChR)激动剂对体外循环(CPB)致大鼠脑损伤时NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体的影响.方法清洁级成年雄性SD大鼠24只,体重350~450 g,进行Morris水迷宫训练5 d后,采用随机数字表法分为3组(n=8):假手术组(S组)、CPB组和CPB+α7nAChR激动剂PHA568487组(CP组).S组不行CPB,只机械通气60 min;CPB组行CPB 60 min;CP组CPB前30 min时腹腔注射PHA5684870.8 mg/kg.术后第3天行水迷宫测试,记录逃避潜伏期和穿越原平台次数.行为学测试后2h时,处死大鼠,取部分海马组织采用TUNEL法测定细胞凋亡情况,计算凋亡指数(AI);其余海马组织采用ELISA法检测IL-1β和IL-18的含量,分光光度法测定caspase-1活性,Western blot法检测NLRP3、凋亡相关斑点样蛋白(ASC)和pro-caspase-1表达水平,RT-PCR法检测NLRP3、ASC和caspase-1的mRNA表达水平.结果与S组比较,CPB组和CP组逃避潜伏期延长,穿越原平台次数减少,海马组织AI、IL-1β和IL-18的含量、caspase-1活性升高,NLRP3和ASC及其mRNA、pro-caspase-1和caspase-1 mRNA的表达上调(P<0.05);与CPB组比较,CP组逃避潜伏期时间缩短,穿越原平台次数增加,海马组织AI、IL-1β和IL-18的含量、caspase-1活性降低,NLRP3和ASC及其mRNA、pro-caspase-1和caspase-1 mRNA的表达下调(P<0.05).结论α7nAChR激动剂减轻CPB致大鼠脑损伤的机制可能与抑制NLRP3炎症小体,减轻脑组织炎症反应有关.

Objective To evaluate the effect of α7 nicotinic acetylcholine receptor (α7nAChR) agonist on inflammasome of NOD-like receptor pyrin domain containing 3 ( NLRP3) during brain injury in-duced by cardiopulmonary bypass ( CPB) in rats. Methods Twenty-four clean-grade adult male Sprague-Dawley rats, weighing 350-450 g, were randomly divided into sham operation group (group S), group CPB, and CPB plusα7nAChR agonist PHA568487 group (group CP) after 5-day Morris water maze train-ing, with 8 rats in each group. Group S was mechanically ventilated for 60 min without receiving CPB. Group CPB received CPB for 60 min. PHA 5684870. 8 mg/kg was intraperitoneally injected at 30 min be-fore CPB in group CP. Water maze test was performed on 3rd day after operation to record the escape laten-cy and times of crossing the original platform. The rats were sacrificed at 2 h after the behavioral test, and their hippocampi were harvested for determination of cell apoptosis ( by TUNEL) and contents of interleukin-1beta ( IL-1β) and IL-18 ( by enzyme-linked immunosorbent assay), caspase-1 activity ( by using spectro-photometry), expression of NLRP3, apoptosis-associated speck-like protein containing a CAR ( ASC) and pro-caspase-1 ( by Western blot or real-time polymerase chain reaction), and expression of NLRP3, ASC and caspase-1 mRNA (using real-time polymerase chain reaction). Apoptotic index (AI) was calculated. Results Compared with group S, the escape latency was significantly prolonged, the times of crossing the original platform were decreased, the AI, contents of IL-1β and IL-18 and caspase-1 activity were in-creased, and the expression of NLRP3 and ASC protein and mRNA, pro-caspase-1 and caspase-1 mRNA was up-regulated in CPB and CP groups (P<0. 05). Compared with group CPB, the escape latency was significantly shortened, the times of crossing the original platform were increased, the AI, contents of IL-1β and IL-18 and caspase-1 activity were decreased, and the expression of NLRP3 and ASC protein and mRNA, pro-caspase-1 and caspase-1 mRNA was down-regulated in group CP (P<0. 05). Conclusion The mechanism by whichα7nAChR agonist alleviates CPB-induced brain injury may be related to inhibiting NLRP3 inflammasome and reducing inflammatory responses in brain tissues of rats.